Michael Palm Basic Science, Vaccines & Prevention Project Weblog

Breakthroughs in Immunology: Free Access Issue of the European Journal of Immunology

"Breakthroughs in Immunology" is the theme of the new issue of the European Journal of Immunology and online access is free of charge. Among the many articles of potential interest:

The macrophage: Past, present and future
Siamon Gordon

Seeing is believing: A focus on the contribution of microscopic imaging to our understanding of immune system function
Marc Bajénoff, Ronald N. Germain

Dendritic cells: Understanding immunogenicity
Ralph M. Steinman

A history of AIDS: Looking back to see ahead
Warner C. Greene

A brief history of CD8 T cells
David Masopust, Vaiva Vezys, E. John Wherry, Rafi Ahmed

NKT cells: In the beginning...
H. Robson MacDonald

HIV Awakens Retroviral Zombies from Cellular Slumber

Back in February the blog covered two conference presentations relating to interactions between HIV and endogenous retroviruses. The gist of the story is that cells infected with HIV appear to also produce fragments of ancient endogenous retroviruses; these remnants of past retroviral infections litter the human genome but cannot replicate or, under normal conditions, transcribe their genetic material into proteins. The HIV protein Vif seems to allow these retroviral zombies to awaken by facilitating the transcription of their genetic material. The production of endogenous retrovirus proteins by HIV-infected cells may thus provide an additional means for the immune system to recognize and target these cells for elimination. Because endogenous retroviruses cannot replicate, they also cannot mutate. So while the rapidly-mutating HIV typically represents a moving target for the immune system,  targeting proteins from endogenous retroviruses may offer an additional, easier means for immune responses to recognize HIV-infected cells.

Keith Garrison and colleagues have now published their data showing that T cell responses to human endogenous retroviruses (HERVs) can be detected in people with HIV and, furthermore, that these T cell responses show a correlation with control of HIV replication during acute HIV infection (the data were also mentioned by senior author Doug Nixon in a recently published interview with TAG). The researchers suggest that the finding may lead to novel HIV vaccine strategies targeting HERVs. However, more study of the circumstances under which otherwise healthy cells might express HERV proteins is needed in order to ascertain the safety and practicality of this idea (so far, HERV expression has been primarily reported in people with HIV and people with certain cancers such as seminoma and breast cancer). A new study looking at this question has also just been published, in the journal AIDS (see second abstract and link, below).

PLoS Pathogens Vol. 3, No. 11, e165 doi:10.1371/journal.ppat.0030165

T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection

Keith E. Garrison1, R. Brad Jones2, Duncan A. Meiklejohn3, Naveed Anwar2, Lishomwa C. Ndhlovu1, Joan M. Chapman1, Ann L. Erickson1, Ashish Agrawal3, Gerald Spotts4, Frederick M. Hecht4, Seth Rakoff-Nahoum5, Jack Lenz6, Mario A. Ostrowski2,7, Douglas F. Nixon1

1 Division of Experimental Medicine, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Immunology, University of Toronto, Toronto, Ontario, Canada, 3 Gladstone Institute of Virology and Immunology, University of California San Francisco, San Francisco, California, United States of America, 4 Positive Health Program, Department of Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America, 5 Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 6 Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America, 7 St. Michael's Hospital, Toronto, Ontario, Canada

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.

AIDS: Volume 21(18), 30 November 2007, p2417-2424

The replicative activity of human endogenous retrovirus K102 (HERV-K102) with HIV viremia

Laderoute MP, Giulivi A, Larocque L, Bellfoy D, Hou Y, Wu HX, Fowke K, Wu J, Diaz-Mitoma F.

OBJECTIVE:: To address the activation and replicative activity of HERV-K102 in vivo associated with HIV viremia. DESIGN AND METHODS:: Initially serology was performed on HERV-K102 specific envelope peptides to determine if HERV-K102 may become activated with HIV viremia. Before developing a quantitative PCR (qPCR) assay, we first determined whether plasma associated particles contained DNA or RNA genomes in a pilot study which surprisingly revealed predominantly DNA genomes. A relative, ddCt qPCR ratio method was then devised to detect excess levels of HERV-K102 pol DNA templates over genomic levels which served as a surrogate marker to reliably index the level of particles found in plasma. RESULTS:: Both the peptide serology and ddCt qPCR excess ratio methods suggested the activation of HERV-K102 in about 70-80% of HIV viremic cases whereas only 2-3% of normal healthy adults had marginally activated HERV-K102 (P < 0.0001). Moreover, by digestion with dUTPase we were able to confirm that the vast majority of excess DNA template in plasma related to cDNA production rather than representing genomic copies. CONCLUSIONS:: Our work uniquely suggests the common activation of HERV-K102 with HIV viremia and may be first to directly demonstrate HERV-K102 cDNA production in vivo. The potential implications of the induction of HERV-K102 activation and replication for the prevention and control of HIV are discussed.

Articles on the Merck HIV Vaccine Trial Results

Several detailed articles regarding the Merck HIV vaccine trial (STEP/HVTN 502) results are now online:

A Step Back? by Kristen Jill Kresge, from the IAVI Report’s November 13 Special Bulletin.

HIV vaccine may have made people more vulnerable to infection by Gus Cairns, from AIDSmap.

Did Merck's Failed HIV Vaccine Cause Harm? by Jon Cohen from Science Magazine (subscription required).

An Eye on the Benign: Pathogenesis Lessons from Non-Pathogenic SIV Infections

The November issue of the Journal of Clinical Investigation (JCI) contains an excellent review of research investigating the benign nature of SIV infection in natural hosts (sooty mangabeys and African green monkeys). Guido Silvestri and colleagues outline current knowledge about the differences between non-pathogenic and pathogenic immunodeficiency virus infections with an emphasis on the now well-accepted role of immune activation in determining disease outcome.

The authors also offer the intriguing suggestion that perhaps non-pathogenic SIV infections have less impact on the function of CD4 T cells because the CD4 T cells that are infected are at a later stage of differentiation and are destined to undergo activation-induced death anyway; in this model, important self-renewing central memory CD4 T cells are not as disrupted by SIV and thus immunodeficiency does not ensue (see image). While speculative, this represents an interesting area for future research.

The authors conclude by stressing that studies of benign SIV infections have made a huge contribution to understanding the pathogenesis of HIV infection in humans, and emphasize that additional studies will likely provide additional important insights, as well as potentially revealing new therapeutic targets for the treatment of the HIV and AIDS. As with all JCI content, access to the full text of the paper is free of charge.

J. Clin. Invest. 117:3148-3154 (2007). doi:10.1172/JCI33034.

Science in Medicine

Understanding the benign nature of SIV infection in natural hosts

Guido Silvestri1,2, Mirko Paiardini1, Ivona Pandrea3,4, Michael M. Lederman4 and Donald L. Sodora5

1Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
2Yerkes National Primate Research Center, Emory University, Atlanta, Georgia, USA.
3Tulane National Primate Research Center, Tulane Health Sciences Center, Tulane University, Covington, Louisiana, USA.
4Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
5Department of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

In striking contrast to HIV infection, natural SIV infection of African nonhuman primates is asymptomatic and usually does not induce significant CD4+ T cell depletion despite high levels of virus replication. Recently, significant progress has been made in understanding the mechanisms underlying the remarkable difference in infection outcome between natural and nonnatural HIV/SIV hosts. These advances include the identification of limited immune activation as a key factor protecting natural SIV hosts from AIDS and the discovery of low CC chemokine receptor 5 expression on CD4+ T cells as a specific and consistent immunologic feature in these animals. Further elucidation of the pathways by which the differences in immune activation between natural and nonnatural hosts are manifest holds promise for the design of novel therapeutic approaches to HIV infection.

Merck HIV Vaccine Trial: Data Presentations from the HVTN Meeting

Laudably, the HVTN has quickly made last Wednesday's data presentations regarding the Merck HIV vaccine efficacy trial results available online:

Open Scientific Session on the STEP Data 

• Introduction to the 502 Protocol, Ann Duerr
• Baseline Characteristics of 3,000 Participants in the STEP Trial, Dan Fitgerald
• STEP Trial: Efficacy Analyses, Mike Robertson
• Exploring hypotheses for differential HIV acquisition rates, Susan Buchbinder
• STEP Study: Summary of Results, Steve Self
• A Pathway Toward Understanding the Biological Basis for the Vaccine Efficacy Results, Danny Casimiro, Julie McElrath
• Roundtable Intro: Follow-up of participants in the STEP Trial
• Longer-Term Follow-Up for STEP/HVTN 502, Peter Gilbert, Steve Self, Devan Mehrotra, Ann Duerr
• Why we unblinded Phambili, Glenda Gray  

Merck HIV Vaccine Trial is Unblinded

It was announced yesterday that the STEP trial will be unblinded; all 3,000 participants will be told whether they received vaccine or placebo and informed of their anti-adenovirus antibody titer.

Merck/HVTN Press Release
San Francisco Chronicle
Baltimore Sun
Seattle Post Intelligencer

Additional Information on the STEP Trial

As mentioned in the previous post, a large amount of information about the Merck HIV vaccine trial was shared yesterday morning at the HVTN conference. Here, in bullet point form, are some more of the potentially important nuggets of information related to yesterday’s presentations:

• Since the September 18 DSMB review, an additional 8 infections have been confirmed in the first 1,500-person STEP cohort (which enrolled people with anti-Ad5 antibody titers less than 1:200).These infections were evenly split between placebo and vaccine recipients, bringing the totals in this cohort to 28 infections among vaccine recipients and 24 among placebo recipients, a non-significant difference.
• HVTN statistician Steve Self explained that, in strict statistical terms, even when the larger difference in infections between vaccine and placebo recipients in the second STEP cohort is factored in, the fact that multiple, unplanned subanalyses are involved means that the difference represents a very strong trend but may not quite reach significance.
• The association between higher levels of anti-Ad5 antibodies and increased susceptibility to HIV infection makes it unlikely that the HIV-specific T cell responses induced by the vaccine were responsible for the observed effect, because HIV-specific T cell responses were of higher magnitude in individuals with low levels of anti-Ad5 antibodies. It also suggests that generalized immune activation induced by the vaccine is not a likely culprit because, again, levels of generalized immune activation would be predicted to be higher in individuals with low levels of anti-Ad5 antibodies. Nevertheless, both of these possibilities will be looked at in detail as part of the plan to evaluate any potential biological mechanism that could explain the study results.
• One potential explanation for the vaccine’s failure to significantly reduce viral load levels in infected participants may be that the vaccine-induced HIV-specific T cell responses were very narrowly targeted. Juliana McElrath showed that recipients developed a T cell response to an average of only one epitope per protein in the vaccine (one epitope from Gag, one from Pol, and one from Nef). Planned analyses will look at the question of whether the epitope-specific T cells could recognize infecting HIV strains and, if so, whether there is evidence that the virus mutated to escape these T cell responses.
• Additional planned immunological and virological analyses include studies of the phenotype (effector memory vs. central memory), functional profiles (e.g. production of various cytokines and proliferative capacity), gene profiles and in vitro antiretroviral efficacy of vaccine-induced T cell responses. A variety of viral sequencing studies in collaboration with James Mullins and Frances McCutchan will also characterize infecting HIV strains and look for evidence of transmission clusters among participants (a phenomenon that was seen in a prior HIV vaccine efficacy trial).
• Among the men in the second STEP cohort who became infected, average set point viral load levels were 19,070 in the 21 vaccine recipients and 89,810 in the 9 placebo recipients. Although this may seem like a large difference, it’s driven by a single individual in the placebo group with a very high viral load and is not significant. These data do, however, offer some reassurance that even if a vaccine-related factor did enhance susceptibility to HIV infection, it did not lead to enhancement of HIV replication among the infected vaccine recipients.
• Susan Buchbinder outlined the three possible non-biological explanations for the STEP results: baseline differences among vaccine and placebo recipients, differential risk behaviors between these groups and simple chance. There were a number of significant baseline differences between the men in the first and second STEP cohorts: more participants in the second cohort were from sites outside the US, of non-Caucasian ethnicity, less than 30 years old, and uncircumcised. However, there were no significant baseline differences between vaccine and placebo recipients in either cohort. Likewise, analyses conducted to date have not found any significant difference in risk behaviors between the vaccine and placebo groups. Buchbinder noted that the small size of the overall study combined with these analyses being restricted to men means that these “post hoc” (unplanned) subset analyses come with very wide confidence intervals, indicating a great deal of uncertainty regarding the robustness of the findings. Given this fact, chance remains a possible explanation of the STEP results, but Buchbinder stressed that the serious implications of the Ad5 vector having an enhancing effect means that it can only be considered as an explanation of exclusion; i.e. all other possibilities must be considered and investigated first.
• At the current time, any STEP participant can ask to be unblinded (told whether they received vaccine or placebo) and informed of their anti-Ad5 antibody titer. Peter Gilbert, a statistician from the HVTN, made the case that maintaining the blind could help show whether any enhancement of susceptibility persisted over time – important information for the long term safety of any recipient of an Ad5 vaccine. Unblinding the study, he argued, could introduce confounding factors that would preclude this analysis. However, other meeting attendees, including Mitchell Warren from AVAC, argued that the blind cannot be ethically maintained if informing participants who received the vaccine might cause them to further reduce their risk of HIV infection. A decision about unblinding the study is expected in a matter of days.
• In a presentation on the unblinding of the Phambili trial, Glenda Gray revealed that the Phambili DSMB met twice – once on September 18, after which they requested additional information about the STEP trial results, and then again on October 8 when they recommended stopping and unblinding the Phambili trial. This appears to contradict recent statements by representatives from the HVTN and Merck that the Phambili DSMB did not review more data than the STEP DSMB.

While the last few weeks have been a nightmare of confusion and uncertainty regarding the STEP and Phambili trials (it is still unclear when the evidence of enhancement among the second STEP cohort came to light), further criticism of the communications strategy employed by the trial sponsors would be redundant and misplaced given the shocking and complex nature of the data. None of the many prior studies conducted with the Ad5 vaccine – in people and in a long series of animal model experiments - have suggested that the presence of anti-Ad5 antibodies could cause the vaccine to increase susceptibility to HIV infection.

Amidst the uncertainty, one thing was very clear from yesterday’s meeting: NIAID, HVTN’s leadership, investigators and collaborators from Merck and elsewhere deserve extraordinary credit for the work they are conducting and planning to figure out what happened in the STEP trial. Both Susan Buchbinder’s and Juliana McElrath’s presentations offered detailed, thoughtful and comprehensive approaches to resolving the questions raised by the study data. The willingness of HVTN and Merck to support outside investigators who might be able to contribute to the effort by suggesting and conducting additional analyses is also to be saluted.

As you’d expect, many more news outlets have now picked up the story and, in general, the coverage is remarkably accurate and well-informed. In hindsight, perhaps this was the trial sponsors justification for initially being economical with the details regarding the data which informed the Phambili DSMB’s decision. Given what had occurred, this may have been the best of a bunch of unpleasant options as to how best to communicate the study findings to the wider public.

Merck HIV Vaccine Trial Results: Uncertainty Reigns Regarding Evidence of Enhancement

The very bad news about the Merck HIV vaccine trial (STEP/HVTN 502) that emerged today at the HVTN meeting in Seattle is that there were significantly more HIV infections among male vaccine recipients in the second 1,500-person cohort of the trial compared to placebo recipients (21 infections vs. 9 infections). The second cohort consisted of individuals with higher levels of antibodies (titers over 1:200) against adenovirus serotype 5 (Ad5), the virus used - in weakened form - as a vehicle or vector for the vaccine components. Ad5 in its natural form causes bad colds, so many people are exposed to it during their lives and develop anti-Ad5 antibodies as a result. The STEP results suggest that the Ad5 vaccine somehow enhanced susceptibility to HIV infection in people with high levels of anti-Ad5 antibodies, as the difference in infection rates between vaccine and placebo recipients increased in parallel with levels of anti-Ad5 antibodies. However, it was also the case that placebo recipients with high levels of anti-Ad5 antibodies had a very low incidence of HIV infection compared to the rest of the STEP cohort, raising the possibility of as yet unexplained confounding effects.

                             

Anti-Ad5 antibody titer	# of infections (vaccine)	# of infections (placebo)
less than 1:18	20	20
1:18-1:200	8	4
1:200-1:1000	14	7
over 1:1000	7	2

                             

Anti-Ad5 antibody titer	HIV incidence (vaccine)	HIV incidence (placebo)
less than 1:18	4%	4%
1:18-1:200	4.4%	2.2%
1:200-1:1000	6.1%	3%
over 1:1000	4.4%	1.2%

These data were not part of the initial review by the DSMB that caused immunizations in the trial to be stopped back in September. Because no women vaccine recipients became infected (only one of the 1,150 women in the trial was infected, a placebo recipient) all of these infections occurred among men. Looking at all of the 1,850 men in both the first and second STEP cohorts, there were 49 infections among the 914 in the vaccine group, compared with 33 out of the 922 in the placebo group. Although this was not a pre-planned analysis, the differences are significant and cause for extreme concern. For the individuals with post-infection viral loads available, there were no significant differences between 46 vaccine recipients (29,109 copies) and 30 placebo recipients (38,416 copies).

As outlined in a whirlwind talk by Juliana McElrath, researchers from the HVTN and Merck have been working around the clock to look at the factors that might account for these observed differences. Some preliminary data indicates that participants with higher anti-Ad5 antibody titers also had higher levels of CD4 T cell activation at week 30 of the study, but this held true in both vaccine and placebo recipients. Whether the kinetics of CD4 T cell activation over time was altered by the vaccine remains to be determined; one hypothesis that has been put forward is that the activation of CD4 T cell responses to Ad5 itself (which would be expected to be of higher magnitude in individuals with higher anti-Ad5 antibody levels) may have contributed to the outcome. McElrath outlined an enormous and comprehensive plan that will look at these and other possible biological mechanisms that might explain the trial results. The plan will involve an expanded version of HVTN’s laboratory science committee (chaired by Bruce Walker) whose task is to further develop and refine the agenda for investigating biological explanations for the STEP results; HTVN will also solicit applications from independent investigators via their website.

In parallel, Susan Buchbinder is evaluating potential behavioral or other non-biological differences between vaccine and placebo recipients in the second STEP cohort. So far, no differences in risk have emerged, although there are some complicated associations with circumcision status; the evidence of enhancement in the trial appears to be skewed toward uncircumcised men but the relevance of this observation, if any, is still unclear.

Researchers at the HVTN meeting are currently discussing how to proceed with follow-up to ascertain whether the increased incidence among vaccine recipients with high anti-Ad5 antibody titers persists over time; 65 of the 82 infections in the trial occurred within a year of the first immunization and those that have occurred since week 52 are roughly evenly split between vaccine and placebo.

TAG will report on the STEP trial results in more detail in the coming days. A number of articles and statements have been issued about today’s announcements:

AVAC on the STEP study: comprehensive links, statement & Q&A
Wall Street Journal article

Doug Nixon Interview

The first in a series of interviews with HIV researchers is now online on the main TAG website. The interview is with Doug Nixon, a highly respected cellular immunologist from UCSF.

This Week’s Merck HIV Vaccine News Round-Up

Late last Friday seems to have seen the peak of the Merck HIV vaccine misinformation frenzy, with the Voice of America managing to get the number of infections in the STEP trial wrong (this article now seems to have disappeared) and a writer for Gay Wired headlining their story “Merck Halts Trials of AIDS Vaccine Shown to Increase Risk of Infection.” The first para of the same article also misrepresents a Seattle Times piece about the trial by claiming it suggests “the vaccine may well increase risk of infection.”

Meanwhile, it seems that it’s now OK to say that the suggestion of enhancement was not based on new data, but the STEP and Phambili DSMB’s interpreting the same data differently; the head of the HVTN, Larry Corey, is quoted to this effect in this week’s issue of Science. The Global HIV/AIDS Vaccine Enterprise has also updated their website to carry the latest news about the STEP and Phambili trials and issued an accompanying statement.

There does still appear to be some cause for concern, however, even though the cause remains unclear. On November 1, the Bay Area Reporter interviewed the STEP principal investigator Susan Buchbinder who, like Tony Fauci in the IAVI Report several weeks ago, makes reference to a “trend” toward enhancement. Since it seems unlikely that either of these individuals would refer to a p value of >0.1 as a trend, the suspicion remains that there is some kind of subgroup analysis where the difference in infections between vaccine and placebo groups comes closer to statistical significance. Things should become somewhat clearer next Wednesday morning, when the data from STEP is scheduled to be presented at a plenary session of the HVTN conference.

In the meantime, some additional details about the STEP trial are available online in a presentation by the HVTN’s Juliana McElrath from the recent IDSA meeting. The demographics of the participants are described, and some data regarding T cell responses 4 weeks after the second immunization (8 weeks into the trial) are presented. What is notable about the T cell response data is that it indicates high numbers of T cells (both CD4 and CD8 together, subsets are not presented) responding to the vaccine-encoded HIV antigens (Gag, Pol and Nef) at this timepoint (~300 spot-forming cells with some individuals >1,000). However, these are almost certainly highly activated T cells as the readout is production of the cytokine interferon-gamma, and prior studies have shown that such high numbers are not maintained at later timepoints. This strongly suggests activation-induced T cell death is still occurring at 4 weeks post-immunization and that most of the HIV-specific T cell responses have not reached a resting central memory state (see prior post on activated vs. resting T cells). CD4 T cell activation sustained for several weeks post-immunization could even be one potential mechanism of enhancement, as some researchers have reported an association between elevated levels of CD4 T cell activation and susceptibility to HIV infection. These issues relating to the phenotype and activation state of the vaccine-induced HIV-specific T cell responses are almost certainly among those that researchers will look at as they try to figure out why the Merck candidate failed.